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Journal of The Lepidopterists^ Society

Volume 13                                            1959                                            Number 4

STUDIES OF THE CHROMOSOMES OF NORTH AMERICAN RHOPALOCERA. 1. PAPILIONIME

by Kodo Maeki and Charles L. Remington

This is the first of a series of papers on the numbers and morphology of the chromosomes of butterflies north of the Isthmus of Panama, with discussions of the phylogenetic significance of the cytological data. The first few papers will include, family by family, material collected in three lots during the 1959 season: 1) around the Rocky Mt. Biological Laboratory in western Colorado; 2) in northwestern Mexico, mainly at Ciudad Victoria (Tamauli-pas) and El Salto (San Luis Potosi) ; and 3) around New Haven, Connecticut. Maeki collected most of the material in Colorado, assisted by Remington and Roger W. Pease, Jr. The Mexican collection was made by Remington. The Connecticut specimens were taken by Maeki, Remington, and Pease. Eric E. Remington brought in important living males in all three regions, and Dr. J. R. Turner took several of the Ciudad Victoria males. New studies will be reported in supplementary papers. Our findings with the cytology of interspecific hybrids will appear elsewhere.

Methods

The techniques will be outlined only in this first paper, because they were used identically for all the families of butterflies which we studied.

The tests provide by far the easiest material in which to investigate the chromosomes of butterflies. First, meiotic divisions exhibit the chromosome complement in haploid condition, so that unequivocal counts of the rather large number of chromosomes are much easier to obtain than in somatic or pre-meiotic divisions. Second, several or many dividing cells can usually be found in the male gonads, whereas female meiosis is limited to the maturation divisions in the egg near the time of entry of the sperm. Third, with Rhopalocera, unlike most other Lepidoptera, male meiosis is usually in process in some cysts of the testes even in old, flown adult males; notable exceptions are the Parnassiinae, Zerynthiinae, Asiatic Graphium, some Hesperi-idae, and Megathymidae, where meiosis is nearly over by the time of eclosion.

193

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The collection of testes is a simple procedure. The testes, which are fused into a single mid-dorsal unit in most Rhopalocera, lie near the dorsal wall of the abdomen about one-half to two-thirds of the distance from the base to the anal end. Wild-caught males are held firmly but unharmed and a small incision is made with sharp, fine scissors in the abdominal wall, over the normal site of the testes. A gentle squeeze of the abdomen is usually sufficient to exsert the testes, with little other tissue emerging. The testes of many species differ strikingly in color from the other abdominal contents and are then readily recognized. They are pinched off with a fine forceps and placed in a vial of fixative. We used Allen's P. F. A. — 3, which has the following formula:

Picric acid —                                   75 cc

Formalin —                                       15 cc

Glacial acetic acid —                     10 cc

Urea —                                                 1 gram

A key number is placed in the vial with the testes. The same number is immediately noted along with locality and date on the paper envelope in which the subsequently killed male is placed for permanent reference. The testes are left in the fixative overnight, for up to 24 hours, and then transferred for storage into a vial of 80% ethyl alcohol.

A few preliminary examinations of freshly collected testes were made by squash preparations in aceto-orcein and aceto-dahlia stain-fixative, but only with species of which we had large samples. All the studies here reported are based on permanent preparations, made at the Gibbs Research Laboratories at Yale University, as follows: the testes were imbedded in paraffin, sectioned at 10// thickness, stained in Heidenhain's iron haematoxy-lin, and counterstained with Light Green. The slides were studied with a Leitz LABOLUX microscope. Suitable nuclei were drawn with camera lucida, with the microscope having 100:X objective and 20X oculars and the drawing set-up giving on the paper a total magnification of 5800X. The photographs were taken with a Leitz MIKAS camera through the above microscope, using the 100X objective and a 10X ocular; the actual magnification on the negative is 333X. One series of counts had to be made primarily from sections of the ovaries of Parnassius smintheus, a species in which we found only a single partially satisfactory division in adult males (see below). The slides and specimens from which the gonads were taken iare being kept for permanent reference in the research series in the Peabody Museum of Natural History of Yale University. The key numbers on these slides and specimens are noted below in square brackets — [ ] and will allow future workers to examine our material.

In addition to the eleven species of Papilionidae described below, we fixed and sectioned testes of 3 Papilio rutulus from Colorado, 2 P. glaucus L. from Connecticut, and 1 P. anchisiades Esper from El Salto but found no dividing cells. The first two species were very old specimens, but the P. anchisiades was obviously a young, fresh male, and it may be that this species normally completes meiosis before eclosion.

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Journal of the Lepidopterists' Society

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Results

1.   Parnassius smintheus Doubleday. The haploid chromosome complement is 7z != 30. This was determined in diploid condition by 6 countable nuclei undergoing oogonial division in the ovaries of a young female with no sphragis (presumably unmated) collected at Copper Lake, el. 10,500', Gunnison Co., Colo., on 7 August [199]. In each of these nuclei the 2n complement of 60 was visible. The apparent differences in size shown in the drawing are probably due to the orientation of the chromosomes and not to actual differences. The base number was verified in haploid condition from one cell in diakinesis in which 30 chromosomes were seen ; this was in a male from Cumberland Pass, 11 August [224]. Testes were also collected from 11 males from Copper Lake (27 July), Cumberland Pass (11 August), and Treasure Mt. (21 August), but no other suitable dividing cells were present. There were many maturation divisions with abnormal meiosis leading to the formation of apyrene sperm. It is usual for Parnassius males to have little or no further normal meiosis after eclosion from the pupal shell. In order to study spermatocyte divisions we will need to collect testes from larvae and pupae. This species has recently been listed under the Palearctic phcebus (Fab.), but we consider this a premature step not justified without biological evidence.

2.   Battus philenor (Linne). The haploid chromosome number is 30. Counts were made in 41 nuclei in 2 males, taken at Ciudad Victoria, Tamps., Mexico, on 1 August [M12] and at El Salto, S. L. P., Mexico, on 4 August [M53]. All suitable nuclei were primary spermatocyte divisions. No normal nuclei at the secondary spermatocyte division were found. The chromosomes of B. philenor are all similar in size.

3.  Papilio ornythion Boisduval. The haploid chromosome number is 30. Counts were made in 15 nuclei in the primary spermatocyte division and 13 nuclei in the secondary spermatocyte division, from 4 males taken at Ciudad Victoria, 2 August [M40-A3, M40-A4, M40-A6, M40-A7]. The size of all the chromosomes is similar. Four other males taken with the above 4 showed no meiotic divisions.

4.  Papilio cresphontes Cramer. The haploid chromosome number is 30. Counts were made in 8 nuclei in the primary spermatocyte division and 3 nuclei in the secondary spermatocyte division, from 2 males taken at Ciudad Victoria, 1 August [Ml 1-2, Ml 1-3], and 15 nuclei in the primary spermatocyte division from a male taken at El Salto, 4 August [M51]. A third male collected at Ciudad Victoria with the first two showed no normal divisions. There appear to be 4 chromosomes distinctly smaller than the other 26; the latter are similar in size. The identifications for P. cresphontes and P. thoas were verified from the male genitalia, which differ widely.

5.  Papilio thoas autocles Rothschild & Jordan. The haploid chromosome number is 27. Counts were made in 36 nuclei, all in the primary spermatocyte division, from a single male taken at Ciudad Victoria, 2 August [M40-B]. Estimating chromosome size can be imprecise due to effects of variation in

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fixation and staining processes and the angle of the sections. Nevertheless, it is clear that only 2 very small chromosomes are present, and we estimate that there are 10 medium-sized, 13 large, and 2 very large chromosomes. Assuming that the ancestral condition for thoas was n = 30 with a caryotype similar to that of cresphontes, it is possible that the evolution of the present caryotype of thoas involved a) fusion of two of the four smallest chromosomes and b) fusions of four of the medium- or large-sized chromosomes to produce the two very large elements. As remarkable as the reduced chromosome number in thoas is the very large size of virtually all of its chromosomes, as compared to primary spermatocyte chromosomes in other Papilio. (See discussion below.) 6. Papilio polyxenes Fabricius. The apparent haploid chromosome numbers are 30 and 31. This is a remarkable situation and resembles the previously known case of Pieris rapa in Japan, discussed below. Four males of P. polyxenes taken in New Haven Co., Connecticut, 23 August to 7 September, gave counts as follows for 32 nuclei:

The thirty-first apparent chromosome is a smaller body than the other 30 and different in its behavior. This element divides during the primary spermatocyte division but somewhat later than do other chromosomes, and it is left out of the nuclei resulting from the second spermatocyte division. In the first division the small element is sometimes in the nucleus, giving a count of 31, and sometimes in the cell outside the nucleus, giving a nuclear count of 30. In some instances it does not appear to divide but at anaphase goes to only one daughter nucleus. (See further discussion below.) Aside from the small element, the chromosomes are all about the same size. No meiotic divisions were found in two other males from Connecticut.

7. Papilio "brucei" Edwards. The nomenclature of this species cannot yet be fixed; a new name may be required. It is the yellow-banded {i.e., zeli-caon-like) Umbelliferae-feeding Papilio of the mountains in Colorado, Wyoming, and probably elsewhere but is not the true lowland zelicaon Boisduval of the West Coast, nor is it the Artemhia-izz&mg middle-altitude true hrucei of Edwards. Genetic studies of this complex will be described soon. The haploid chromosome number is 30. Counts were made in 5 nuclei of a single male taken at Gothic, Gunnison Co., Colorado, on 16 July [1]. No secondary spermatocyte divisions were found. The chromosomes are all similar in size. The testes are unusual in this species. They are connected only slightly, whereas the testes of other Papilioninae are so broadly joined that the pair appears as a single ovoid unit.

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Journal of the Lepidopterists' Society

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"m

?*$^

i

,,§§

Jl %

ife

*#•*>

4e

•••■•

.^ipi

Jill

^%V««*

SliKMS"

-KT■: ' - v-i:

JHi£

Photographs of chromosomes in spermatocyte divisions. Fig. 1 — Battus philenor (I) ; fig. 2 — Papilio cresphontes (I) ; fig. 3 — P. thoas (I) ; figs. 4a-c — P. polyxenes; fig. 5 — P. "brucei" (I) ; fig. 6 — P. palamedes (I) ; fig. 7 —-P. pilumnus (I).

8.  Papilio troilus Linne. The haploid chromosome number is 30. Counts were made in 6 nuclei in the primary spermatocyte division and 7 nuclei in the secondary spermatocyte division, all from one male taken at West Rock, New Haven Co., Connecticut, 24 August [390]. All the chromosomes are similar in size. No meiotic divisions were found in a second male collected on 3 September.

9.   Papilio palamedes Drury. The haploid chromosome number is 30. Counts were made in 7 nuclei, all in the primary spermatocyte division, from 2 males taken at Ciudad Victoria, 1 August [M9-1, M9-2]. Two chromosomes are distinctly larger than the others and some were much smaller than the several medium-sized elements.

10.  Papilio pilumnus Boisduval. The haploid chromosome number is 30. Counts were made in 8 nuclei in the primary spermatocyte division and 9 nuclei in the secondary spermatocyte division, all from one male taken at Ciudad Victoria, 2 August [10-2]. The size and form of all the chromosomes are similar. No meiotic divisions were found in a second male taken the same dav.

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11. Graphium phaon (Boisduval). The haploid chromosome number is 30. Counts were made in 5 nuclei in 1 male, taken at Ciudad Victoria on 2 August [M41-2]. These nuclei were all in the primary spermatocyte division. The chromosomes are all similar in size (compare with Japanese Graphium discussed below). It is of unusual interest that spermatocyte divisions were present in a flying {i.e., imaginal) individual of G. phaon. In contrast, G. sarpedon and G. doson in Japan are like Parnassiinas, Zeryn-thiinas, and Megathymidae in having meiosis essentially complete in the male prior to eclosion. The degree of proximity of New World and Oriental-In-doaustralian so-called Graphium is somewhat doubtful on grounds of gross morphology, and the chromosome size and meiotic timing do not strengthen the case for lumping the whole array in a single genus along with the typical Iphiclides Kite Swallowtails.

Discussion

The latest atlas of chromosome numbers (Makino, 1956) lists published counts for 197 species of Rhopalocera (butterflies) and 194 species of the remaining Lepidoptera (moths), including some corrections of taxonomic listing made by us. Several of these counts, especially by early workers, are surely incorrect, but additional counts have been recorded since 1956 by Saitoh, de Lesse, Lorkovic, and Maeki and the actual number of valid counts is probably about 200 for butterflies and 200 for moths. There are now about 130,000 described species of Lepidoptera of which about 10% are Rhopalocera. Undoubtedly relatively few new Rhopalocera remain to be named, whereas there may be as many unnamed as named moths. So it is obvious that cytotaxonomy of moths is hardly started, and even for butterflies only a preliminary sampling has been made (and that only for the Holarctic region).

Table 1 shows the chromosome numbers of the 32 species of Papilionidae for which counts have been recorded. The 11 species described in the present paper are all new to cytology and are shown in capitals in the Table. For G. podalirius there is also a count of 54 to 58 at the diploid stage by Kernewitz (1915), but this, considered an error in view of the precise haploid counts of 30 at both spermatocyte divisions by Lorkovic (1941). Munson (1907) reported for P. rutulus 2n >= 28 during spermatogenesis, but his Figure 89 clearly shows 32 in a "dividing spermatocyte." It will be surprising if P. rutulus differs much from n *= 30. We collected testes from badly worn males in Colorado but failed to find any normal meiotic stages, and we therefore cannot yet correct or verify the Munson haploid counts. The diploid number is obviously wrong.

The Table shows how stereotyped the chromosome number is for the Papilioninae, 30 being a rule seldom violated. Our discovery of n *= 27 for P. thoas is remarkable in view of the frequency of n = 30, even in the sibling of thoas, P. cresphonteSj and their near relative P. ornythion. The presence of unusually large chromosomes in P. thoas strongly suggests derivation of

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its reduced number by fusion of 6 of the ancestral elements into 3 thoas chromosomes, producing increased polyteny as well as actual length. Cyto-photometric determinations of DNA might show whether this in fact occurred (Hughes-Schrader & Schrader, 1956). It will be important to examine the chromosomes of geographically distant populations of cresphontes and thoas {e.g., pennsylvanicus F. & R. Cherm. and brasiliensis R. & J.) to see whether the formulae n ^= 27 and n = 30 are consistent species differences. N — 27 is not the lowest known formula for a papilionid; Graphium sarpedon has only 20, all large, ten of these probably derived by 1 + 1 fusion of 20 of the small ancestral chromosomes such as are now present in the closely related G. doson (doson has 20 small and 10 large chromosomes).

The extra element in P. polyxenes is our most notable finding in these new Papilionidae. We intend to trace it in FL hybrids with species not normally having the element. Maeki (1959) had shown that the Japanese so-called race of Pieris rapsE L. (subspecies crucivora Bdv.) has an extra element, plus 25 normal chromosomes. This extra element has characteristics of Wilson's "m-chromosome" (1925: p.839). The m-chromosome is very small and is sometimes present in the nucleus (n == 26) and sometimes in the extra-nuclear cytoplasm (n := 25). The European and American populations of P. rap<% lack the m-chromosome entirely. Pieris melete Men. of Japan has a base number of n'^27, and as many as 4 minute supernumeraries may be present in the nucleus. From 0 to 2 supernumeraries may also be found in nuclei of Melanitis phedima (Cramer), a Japanese satyrid with a base complement of n != 28. The only Papilio with a regular complement of n c= 31 is P. hippocrates (Maeki, 1953) ; in this species all the chromosomes are similar in size, and the indistinguishable 31st chromosome does not appear to be similar to a supernumerary or m-chromosome.

Summary

1.   Chromosome counts are presented for eleven species of Papilionidae, all new to cytology. This brings to 32 the number of species of Papilionidae for which chromosome counts are recorded. The new counts are for Par-nassius smintheus (Colorado), Battus philenor (Mexico), Papilio ornythion (Mexico), P. cresphontes (Mexico), P. thoas (Mexico), P. polyxenes (Connecticut), P. "brucei" (Colorado), P. troilus (Connecticut), P. palamedes (Mexico), P. pilumnus (Mexico), and Graphium phaon (Mexico). The Parnassius count is primarily from diploid oogonial nuclei; adult males showed no meiotic divisions. All the Battus, Papilio, and Graphium counts are from primary and/or secondary spermatocyte divisions (haploid) in testes of adult males taken in the wild.

2.   Graphium phaon differs from G. doson and G* sarpedon of Japan in having meiotic divisions in the adult male and in having all the chromosomes similar in size.

1959                                          Journal of the Lepidopterists' Society                                         201

3. The count for Parnassius smintheus is 2n *=■ 60. Eight of the ten Papilioninae consistently have n *= 30. One exception is P. polyxenes with rc = 30 and 31 in primary spermatocyte divisions due to the common but not invariable presence of an additional irrnute element (m-chromosome) which is sometimes extra-nuclear in position. The count for P. thoas of n t= 27 is the more noteworthy since its sibling species P. cresphontes has n t= 30, like 14 of the 15 other Papilio species reliably reported. (The other exception is P. hippocrates of Japan with n i= 31.) The relative sizes within the complement suggest that the n = 27 set is derived, from an ancestral set with n — 30, by fusion of two tiny chromosomes and 1 + 1 fusions of four larger chromosomes. Another difference is that most of the chromosomes of thoas are much larger than in cresphontes and other species of Papilio.

Acknowledgements

We are grateful to the collectors noted on the first page and to P. Sheldon Remington, Don B., Viola T., and Jack Stallings for field assistance in Mexico, to Dr. and Mrs. Ross Dickson of Ciudad Victoria for excellent facilities, and to Dr. R. Bruce Nicklas of the Department of Zoology of Yale University for discussing the manuscript. This work was supported in all its parts by a research grant (G 3830) from the U. S. National Science Foundation.

References

Federley, H., 1938. Chromosomenzahlen finnlandischer Lepidopteren. I. Rhopalocera. Hereditas 24: 397-464, 47 figs.

Hughes-Schrader, S., & F. Schrader, 1956. Polyteny as a factor in the chromosomal evolution of the Pentatomini (Hemiptera). Chromosoma 8:35-151, 5 figs.

Kernewitz, B., 1915. Spermiogenese bei Lepidopteren mit besonderer Berucksichtigung der Chromosomen. Arch. Naturgeschichte {A) 81: 1-34, 3 pis., 14 figs.

Lorkovic, Z., 1941. Die Chromosomenzahlen in der Spermatogenese der Tagfalter. Chromosoma 2: 155-191, 13 figs.

Maeki, K., 1953. Chromosome number of some butterflies (Lepidoptera-Rhopalocera). Jap. journ. genetics 28: 6-7, 5 figs.

............, 1957. A cytological study in 16 species of the Japanese Papilionidae (Lepidoptera-Rhopalocera) [in Japanese]. La Kromosomo 32: 1115-1122, 29 figs.

............, 1959. A chromosomal study in fifteen species of the Japanese Pieridae (Lepidoptera-Rhopalocera). Kvuansei Gakuin Univ. annual studies 7: 361-368, 32 figs.

Makino, S., 1956. A review of the chromosome numbers in animals, rev. ed. 300 pp. Hokuryukan, Tokyo.

Munson, J. P., 1906. Spermatogenesis of the butterfly, Papilio rutulus. Proc. Boston soc. nat. hist. 33: 43-124, pls.12-17.

Regnart, H. C, 1930. Additions to our knowledge of chromosome numbers in the Lepidoptera. Proc. Univ. Durham philos. soc. 9: 79-83, figs.

Wilson, E. B., 1925. The cell in development and heredity, 3rd ed. 1232 pp., 529 figs. Macmillan, New York.

Department of Zoology, Yale University, New Haven, Conn., U. S. A.

PLATE 1

CHROMOSOMES

• ••

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• ••

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13b                                       14b       • • •

Fig. 8 — Parnassius smintheus oogonial division; fig. 9 — Battus philenor (I) ; fig. 10a — Papilio ornythion (I) ; fig. 10b — same (II) ; fig. 11a — P. cresphontes (I) : fig. lib — same (II) ; fig. 12 — P. thoas (I) ; fig. 13a — P. pilumnus (I) ; fig. 13b — same (II) ; fig. 14a — P. troilus (I) ; fig. 14b — same (II); fig. 15 — P. palamedes (I) ; fig. 16 — Graphium phaon (I). I t= primary spermatocyte division; II t= secondary spermotocyte division. Magnifications on Plates 1 and 2 all 3900 diameters.

CHROMOSOMES

PLATE 2

••••• • ••• • •

j ••••

17 •••

18a

•   • •

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18c

18d

•••••••

m

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19a

19c

Fig. 17 — Papilio "brucei" (I) ; figs. 18a-20 all P. polyxenes: fig. 18a — (I), no m-chromosome; fig. 18b — (II), no m-chromosome; fig. 18c — (I), m-chromosome as regular member of complement; fig. 18d — (I), m-chromosome in cytoplasm; fig. 19a— m-chromosome unpaired at metaphase plate; fig. 19b — minute element not certainly recognizable; fig. 19c — m-chromosome outside nuclear spindle; fig. 20 — m-chromo-somes dividing much later than regular chromosomes.